Nucleic acid amplification has proven useful in numerous clinical applications including the detection and/or diagnosis of infectious diseases and pathological genomic abnormalities as well as nucleic acid polymorphisms that may not be associated with any pathological state. Nucleic acid amplification is particularly useful in circumstances where the quantity of the targeted nucleic acid is relatively small compared to other nucleic acids present in a sample, where only a small amount of the targeted nucleic acid is available, where the detection technique has low sensitivity, or where more rapid detection is desirable. For example, infectious agents may be accurately identified by detection of specific characteristic nucleic acid sequences. Because a relatively small number of pathogenic organisms may be present in a sample, the nucleic acid extracted from these organisms typically constitutes only a very small fraction of the total nucleic acid in the sample. Specific amplification of the characteristic nucleic acid sequences, if present, greatly enhances the sensitivity and specificity of the detection and discrimination processes.
Generally, the currently known amplification schemes can be broadly grouped into two classes based on whether the enzymatic amplification reactions are driven by continuous cycling of the temperature between the denaturation temperature, the primer annealing temperature, and the amplicon (product of enzymatic amplification of nucleic acid) synthesis temperature (thermocycling amplification), or whether the temperature is kept constant throughout the enzymatic amplification process (isothermal amplification). Typical cycling nucleic acid amplification technologies (thermocycling) are polymerase chain reaction (PCR), and ligase chain reaction (LCR). FSpecific protocols for such reactions are discussed in, for example, Short Protocols in Molecular Biology, 2nd Edition, A Compendium of Methods from Current Protocols in Molecular Biology, (Eds. Ausubel et al., John Wiley & Sons, New York, 1992) chapter 15. Reactions which are isothermal include: transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), and strand displacement amplification (SDA), among others.
Isothermal target amplification methods include transcription-based amplification methods, in which an RNA polymerase promoter sequence is incorporated into primer extension products at an early stage of the amplification (WO 89/01050), and further target sequence, or target complementary sequence, is amplified by transcription steps and digestion of an RNA strand in a DNA/RNA hybrid intermediate product. See, for example, U.S. Pat. Nos. 5,169,766 and 4,786,600. These methods include transcription mediated amplification (TMA), self-sustained sequence replication (3SR), Nucleic Acid Sequence Based Amplification (NASBA), and variations there of. See, for example, Guatelli et al. Proc. Natl. Acad. Sci. U.S.A. 87:1874-1878 (1990); U.S. Pat. Nos. 5,766,849 5,399,491; 5,480,784; 5,766,849; and 5,654,142 (TMA); U.S. Pat. No. 5,130,238 (Malek et al.); U.S. Pat. No. 5,409,818 and EP0329822 (Davey et al.); U.S. Pat. No. 5,654,142 (Kievits); and U.S. Pat. No. 6,312,928 (Van Gemen et al.) (nucleic acid sequence-based amplification (NASBA) techniques); and Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177; Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878; PCT Patent Publication No. WO 92/08800) (3SR). U.S. Pat. No. 5,744,311 (Fraiser); U.S. Pat. No. 5,648,211 (Fraiser); U.S. Pat. No. 5,455,166 (Walker) and U.S. Pat. No. 5,631,147 (Lohman), describe isothermal amplification systems based on strand displacement amplification (SDA).
DNA NASBA methods (DNA target amplification resulting in RNA amplicons) have also been described ((see, e.g., ‘Method for the amplification and detection of DNA using transcription based amplification’ (WO 02/070735), ‘Method for the amplification and detection of HBV DNA using transcription based amplification’ (WO 02/072881) and ‘Nucleic acid sequences that can be used as primers and probes in the amplification and detection of HSV DNA and method for the amplification and detection of HSV DNA using transcription based amplification’ (EP04078166.8)).
U.S. Pat. Nos. 4,683,195 and 4,683,202 provide description of PCR. U.S. Pat. No. 5,792,607 (Backman) describes amplification methods referred to as ligase chain reactions (LCR; see also Wu and Wallace, 1989, Genomics 4:560-569 and Barany, 1991, Proc. Natl. Acad. Sci. USA 88:189-193). Other approaches include Polymerase Ligase Chain Reaction (Barany, 1991, PCR Methods and Applic. 1:5-16); Gap-LCR (PCT Patent Publication No. WO 90/01069); Repair Chain Reaction (European Patent Publication No. 439,182 A2); Q-beta replicase, transcription mediated iso CR cycling probe technology, and cascade rolling circle amplification (CRCA). Additional U.S. patent documents which describe nucleic acid amplification include U.S. Pat. Nos. 4,876,187; 5,030,557; 5,399,491; 5,485,184; 5,554,517; 5,437,990; 5,399,491, 5,554,516 and 6,551,778. A survey of amplification systems is provided in Abramson and Myers, 1993, Current Opinion in Biotechnology 4:41-47.
In a standard NASBA reaction, large amounts of single stranded RNA are generated from either single stranded RNA (ssRNA) or DNA (ssDNA) or double stranded DNA (dsDNA) (U.S. Pat. No. 5,654,142). When RNA is to be amplified with NASBA the ssRNA serves as a template for the synthesis of a first DNA strand by elongation of a first primer containing a RNA polymerase recognition site. This DNA strand in turn serves as the template for the synthesis of a second, complementary, DNA strand by elongation of a second primer, resulting in a double stranded active RNA-polymerase promoter site, and the second DNA strand serves as a template for the synthesis of large amounts of the first template, the ssRNA, with the aid of a RNA polymerase (U.S. Pat. No. 5,409,818).
Transcription based amplification techniques involve the transcription of multiple RNA copies from a template comprising a promoter recognized by an RNA polymerase. With these methods multiple RNA copies are transcribed from a DNA template that comprises a functional promoter recognized by the RNA polymerase. These copies are used as a target again from which a new amount of the DNA template is obtained etc. Isothermal transcription based amplification can be performed (Davey et al., EP 323822 (NASBA); Gingeras et al., EP0373960; Kacian et al., EP0408295), and also transcription based amplification reactions may be performed with thermostable enzymes which allow the reaction to be carried out at more elevated temperatures (e.g., EP0682121, Toyo Boseki K K).
The methods as described in EP0323822, EP0373960 and EP0408295 are isothermal continuous methods. With these methods four enzyme activities are required to achieve amplification: an RNA dependent DNA polymerase activity, an DNA dependent DNA polymerase activity, an RNase (H) activity and an RNA polymerase activity. Some of these activities can be combined in one enzyme, so usually only two or three enzymes are necessary. Enzymes having RNA dependent DNA polymerase activities are enzymes that synthesize DNA from an RNA template. A DNA dependent DNA polymerase thus synthesizes DNA from a DNA template. In transcription-based amplification reactions a reverse transcriptase such as AMV (Avian Myoblastosis Virus) or MMLV (Moloney Murine Leukemia Virus) reverse transcriptase may be used. Such enzymes have both RNA- and DNA dependent DNA polymerase activity but also an inherent RNase activity. In addition an RNase may be added to the reaction mixture of a transcription based amplification reaction, such as E. coli RNase H.
Conventional techniques of molecular biology and nucleic acid chemistry, which are within the skill in the art, are fully explained in the literature. See, for example, Sambrook et al., 1989, Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins. eds., 1984); and a series, Methods in Enzymology (Academic Press, Inc.). Nucleic acid hybridization techniques have been described for example, in Sambrook et al.; U.S. Pat. No. 4,563,419 (Ranki) and U.S. Pat. No. 4,851,330 (Kohne) and in Dunn et al., Cell 12, pp. 23-26 (1978) among many other publications.
Detection methods utilizing nucleic acids are also known. Nucleic acids are often labeled for various detection purposes. For example, methods described in U.S. Pat. No. 4,486,539 (Kourlisky); U.S. Pat. No. 4,411,955 (Ward); U.S. Pat. No. 4,882,269 (Schneider) and U.S. Pat. No. 4,213,893 (Carrico), illustrate preparation of labeled detection probes for detecting specific nucleic acid sequences. Furthermore, before or after exposing an extracted nucleic acid to a probe, the target nucleic acid can be immobilized by target-capture means, either directly or indirectly, using a “capture probe” bound to a substrate, such as a bead or a magnetic bead. Examples of target-capture methodologies are described by Ranki et al., U.S. Pat. No. 4,486,539, and Stabinsky, U.S. Pat. No. 4,751,177. Further uses of probes have been described, for example, in U.S. Pat. Nos. 5,210,015; 5,487,972; 5,804,375; 5,994,076.
Additionally, a class of oligonucleotide probes, referred to as molecular beacons, that facilitate homogeneous detection of specific nucleic acid target sequences has been described (Piatek et al. (1998) Nature Biotechnology 16:359-363; Tyagi and Kramer (1996) Nature Biotechnology 14:303-308). Molecular beacons are nucleic acid sequences that contain a fluorophore and a quencher. By design, molecular beacons are expected to fold into stem-loop structures in which the fluorophore is placed in close proximity to the quencher. When the molecular beacon is illuminated with light corresponding to the excitation wavelength of the fluorescent group, no fluorescence is observed, because energy transfer occurs between the fluorescent group and the quenching group, such that light emitted from the fluorescent group upon excitation is absorbed by the quenching group. The loop region of molecular beacons is designed to contain a nucleotide sequence complementary to the target sequence of interest. When the molecular beacon is contacted with sequences complementary to the loop, the loop hybridizes to this sequence. This process is energetically favored as the intermolecular duplex formed is longer, and therefore more stable, than the intramolecular duplex formed in the stem region. As this intermolecular double helix forms, torsional forces are generated that cause the stem region to unwind. As a result, the fluorescent group and the quenching group become spatially separated such that the quenching group is no longer able to efficiently absorb light emitted from the fluorescent group. Thus, binding of the molecular beacon to its target nucleic acid sequence is accompanied by an increase in fluorescence emission from the fluorescent group. Molecular beacons undergo intermolecular hybridization upon interaction with the specific target sequence. Molecular beacons have been used for homogeneous detection of specific nucleic acid sequences, both DNA and RNA (Leone et al. (1998) Nucleic Acids Research 26:2150-2155; Piatek et al. (1998); Tyagi and Kramer (1996)).
A number of target nucleic acids have proven difficult to amplify to readily detectable levels and/or with appropriate specificity (e.g., HCV (Pavio and Lai, J. Biosci. 28(3): 287-304 (2003)), among others), and methods to improve amplification are thus needed. Additionally, improvements to amplification can be useful to any target for which faster results are desired. Furthermore, some targets have proven difficult to amplify when in a multiplex reaction with other targets and/or primers and probes specific for other targets.
Some improvements in amplification have been made. U.S. Pat. No. 6,338,954 (van Gemen) discloses a method for non-specific amplification. U.S. Pat. No. 6,509,157 discloses amplification primers used in PCR reversibly blocked such that primer is unblocked at temperatures used to start PCR. U.S. Pat. No. 5,849,497 discloses a method for inhibition of amplification of at least one target sequence in a PCR reaction.
Thus, while many advances have been made in the area of amplification of nucleic acids, there is still a need to improve the product yield, to achieve improved sensitivity for a specific target and thus to provide more useful assays, particularly when reagents specific for more than one target are present, such as in a multiplex assay, and even more particularly, when amplification of one target in a multiplex assay dominates over amplification of a second target in the assay. The present invention provides methods to improve transcription-based amplification of a specific target that can be applied to many selected target nucleic acids and combinations thereof.